Insulin resistance (IR) and hyperinsulinemia are important cardiovascular risk factors in diabetic patients. Insulin actions have been identified on endothelial cells (EC), vascular smooth muscle cells (VSMC) and macrophages. Yet, the question of whether insulin has pro- or anti-atherogenic actions has been hotly debated for many years. We propose that the acceleration of atherosclerosis in diabetes is due to selective insulin resistance in the vascular wall where hyperglycemia and elevated free fatty acids (FFA) can inhibit insulin signaling through the IRS/PI3K/Akt pathway. This may decrease insulin's anti-atherogenic actions in EC such as activation of eNOS, increased expression of heme oxygenase-1 and decreased expression of VCAM-1. Also, insulin and PKC activation may enhance the MAPK pathway leading to increased expression of pro- atherogenic activities such as ET-1, PAI-1 expression, migration and proliferation of VSMC, creating selective insulin resistance in the vessel wall. In this grant period, we showed that deletion of insulin receptors, specifically in EC, accelerated atherosclerosis due to the loss of novel insulin actions to decrease VCAM-1 expression. We also showed that hyperinsulinemia alone, without vascular or systemic IR, did not accelerate atherosclerosis in ApoE-/- mice. We have shown that one cause of selective insulin resistance in the endothelium is due to the activation of PKC, especially of the a and b isoforms, induced by hyperglycemia and elevated FFA. We also identified the targets of PKCb activation induced by angiotensin on insulin signaling molecules including p-Ser303 in IRS2 and p-Thr86 of p85a/PI3K. The overexpression of PKCb isoform, specifically in EC, selectively inhibited insulin signaling through the IRS/Akt pathway and exacerbated atherosclerosis. Thus, we propose to: 1. Characterize the effect of exogenous insulin treatment and PKCb inhibition on the development of atherosclerosis in a new model of ApoE-/- mice, which exhibit hyperinsulinemia, hyperglycemia, hypercholesterolemia and IR at the vessel wall, and systemically by feeding a high fat diet (HFD, 60% fat) instead of a western diet (WD, 42% fat), providing a simple diet induced model with atherosclerosis that mimics all of the metabolic abnormalities of type 2 diabetes. 2a. Evaluate ApoE-/- mice overexpressing IRS1 specifically in the endothelium, EIRS1/ApoE-/- mice, which showed dramatic decreases in atherosclerosis, and possibly identified a novel pathway by which insulin activates eNOS via increases in endothelin B receptor (ETB) expression by a pathway independent of p-S(1176)eNOS. 2b. Determine whether intimal hyperplasia and atherosclerosis will decrease in mice which are expressing S-A(1176)eNOS and overexpressing IRS1 in EC on ApoE-/- background while on HFD. 3. We will address the question of insulin's potential atherogenic effects on VSMC by determining the role of insulin and its receptor on aortic SMC regarding their impact on atherosclerotic lesions in ApoE-/- mice with deletion of insulin receptor on HFD.